BIOCHEMISTRY
A Method for Locating 4-Thiouridylate in the Primary Structure of Transfer Ribonucleic Acids* Edward B. Zifft and Jacques R. Fresco
ABSTRACT : Transformation of 4-thiouridylate residues of transfer ribonucleic acid to radioactively labeled N4-methylcytidylate residues is achieved under mild conditions where the major bases are unaffected and the polymeric structure of transfer ribonucleic acid remains intact. The introduction of this unique, chemically stable, radioactive label at the site of 4-thiouridylate enables the use of standard sequence methods
A
lthough general procedures are available for the determination of the primary structures of tRNAs (cJ: Holley et a/., 1965; Sanger et a/., 1965), specific problems arise in locating particularly labile minor nucleotides (cJ: Madison, 1968). Among these labile residues is 4-thioU,l a constituent of Escherichia coli tRNA (Lipsett, 1965), which is subject to desulfuration under some conditions of tRNA fractionation (cf. Weeren et al., 1969) and of sequence analysis (Madison, 1968). In the course of investigating chemical transformations of 4-thioU residues, reactions have been developed which can incorporate a chemically stable and unique radioactive (I4C or 3H) label at the site of 4-thioU in the primary structure of tRNA. The labeled product of this transformation, N4-mC, may be readily located by established sequencing techniques. The reactions employed for this transformation were previously elaborated for thionucleoside model compounds (Ziff and Fresco, 1968). Under appropriate conditions, oxidation of the 4-thiouracil moiety of these nucleosides by sodium periodate (cf. Scheme I) yields the corresponding 2-oxypyrimidine-4-sulfonate nucleosides. The latter compounds are highly reactive at the 4 position with a variety of oxygen, nitrogen, and sulfur nucleophiles; hydrolysis at acid or alkaline pH yields uracil nucleosides, while ammonolysis with N H 3 or CH3NHr yields cytosine and N4-methylcytosine nucleosides, respectively.
* From the Program in Biochemical Sciences, Frick Chemical Laboratory, Princeton University, Princeton, New Jersey 08540. Receiced April 8, 1969. Supported by grants from the National Institutes of Health (GM-07654), the National Science Foundation (GB-6664), and the American Heart Association. t U. S . Public Health Service predoctoral fellow (1964-1968) and predoctoral trainee (1968-1969) (Grant 5T01-GM00962). 1 Abbreviations used are: free nucleotides are indicated by the letters M P (monophosphate) preceded by the letter symbol for the base moiety, in turn preceded by the number indicating site of ribose esterification, e.g., 2'43 ')-4-thioUMP = 2'(3 ')-4-thiouridylate; nucleotide residues of a nucleic acid are symbolized either by the letter symbol of the base moiety, e.g., N4-mC = N4-methylcytidylate residue, or in conjunction with the word residue; PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis[2-(5-phenyloxazolyl)]benzene;R, = ratio of distance traveled by compound to that traveled by reference compound in electrophoresis.
3242
ZIFF
A N D FRESCO
for locating this unstable residue in the primary structure d transfer ribonucleic acid. Using this labeling procedure, the distribution of 4-thiouridylate among the T-1 ribonuclease digestion products of unfractionated Escherichia coli B transfer ribonucleic acid has been scanned, and the sequence around the 4-thiouridylate residue of E. coli B valine transfer ribonucleic acid has been investigated.
In the present work this reaction sequence has been used to transform 4-thioU in tRNA to labeled N4-mC. Under the conditions employed, the major bases are unreactive, and the polymeric structure of tRNA remains intact. The ability to introduce this unique and stable radioactive label at the site of 4-thioU has been exploited for the purpose of locating this residue in the oligonucleotides obtained from RNase T-1 digests of unfractionated E. coli B tRNA, and a purified tRNAVa'from E. coli B. Materials Unfractionated E. coli B tRNA was obtained from Schwarz BioResearch, Inc. It was dialyzed (4') exhaustively US. 1.0 M NaC1-0.01 M EDTA (pH 7.5) and then cs. H 2 0 , and then was lyophilized. Purified E. coli B tRNAVa1(major valine-acceptor tRNA) was the generous gift of Dr. A. D. Kelmers. It accepted 1.2 nmoles of valine/ilj60 unit, and had A260/A335 ,40. The preparation was exhaustively dialyzed (4') L'S. 0.01 M EDTA (pH 7 . 9 , then cs. H 2 0 , and was then lyophilized. Unfractionated baker's yeast tRNA was prepared as previously described (Lindahl and Fresco, 1967). Alkaline phosphatase, RNase A, and snake venom phosphodiesterase were obtained from Worthington Biochemical Corp. RNase T-1 was purified by a modification of the methods of Takahashi (1961) and Rushizsky and Sober (1962). [ 14C]Methylamine-HC1 was obtained from Nuclear Chicago Corp., and utilized at 0.78 Ci/mole unless otherwise noted. Methylamine-HC1 (Matheson-Coleman, recrystallized once from H20) was warmed in NaOH solution in a gasgenerating apparatus, and the resulting gas was bubbled through freshly boiled distilled HLO. The concentration of methylamine solutions so obtained was determined by HCI titration to the methyl red end point. The pH of solutions of methylamine was adjusted with HCl. Solutions of sodium periodate (Fisher, reagent grade) were prepared just prior to use. Cacodylic acid (Fisher) was recrystallized from H1Oethanol solution to remove ultraviolet-absorbing impurities. N4-Methylcytidine was prepared as previously described (Ziff and Fresco, 1968). Deoxyribonucleosides were obtained from CalBiochem; Sephadex G-100 and Sephadex A-25 from
V O L . 8,
NO.
8,
AUGUST
1969
Pharmacia Fine Chemicals; Dowex AG 1-X4 from Bio-Rad Corp. ; and Whatman DE-32, microgranular DEAE-cellulose from Reeve-Angel, Inc. The latter was washed and equilibrated with buffer according to manufacturer's directions. Paper for chromatography was Whatman No. 1, used as obtained.
SCHEME I
Methods Ultruciolet Absorption. Measurements were performed with either a Cary Model 14 spectrophotometer, using 1.0 and 0.1 slide wires as required (spectra), or with a Beckman D U monochromater equipped with a Gilford Model 220 optical density converter (single wavelength measurements). Radioacticity. A Packard scintillation spectrometer was employed. Aqueous solutions were counted in 15 ml of Bray's solution (Bray, 1960), while samples retained on Millipore filters or on paper chromatogram strips were counted in a scintillation fluid containing 4 g of PPO and 200 mg of POPOP per 1. of toluene. Counting efficiency was determined either by the channels ratio technique or by counting a known number of micromoles of [14C]methylamine under standard conditions. tRNA Concentration. This was determined spectrophotometrically in 0.01 M MgCln-0.01 M cacodylate (Na+) (pH 7.0), using the relationships: A260 0.1 solution tRNA = 20.5 (Lindahl and Fresco, 1967) 1.8 nanomoles of tRNA = 1 unit. Dericatization of Unfractionuted tRNA. tRNA (1.5 mg/ml) in 0.0078 M [14C]methylamine (pH 10.4)-0.0078 M N a I 0 4 (added last to initiate reaction) was incubated at 40" for 45 min. The tRNA was precipitated with 0.05 volume of 3 M NaCl and 2 volumes of ethanol at 0' (1 hr), collected by centrifugation, and dialyzed (4') cs. 0.01 M NaCI-0.01 M phosphate (Na+), pH 7 (400 volumes, 2 changes), and then exhaustively es. H 2 0 . The course of this reaction was determined by withdrawing aliquots (100 p l ) at designated times, and precipitating the tRNA at 0" with 1.5 ml of 10% trichloroacetic acid. The precipitate was collected on Millipore filters, rinsed with trichloroacetic acid, and counted. Iodine-Oxidized tRNA. E. coli B tRNA (7 mg/ml) in 0.002 M Is-0.002 M KI-0.01 M phosphate (Na+, pH 7.0) was incubated for 30 min at 0". The tRNA was freed of reagents by repeated precipitation with NaCl and ethanol, dialysis (4') cs. 800 volumes of 0.15 M NaCI-0.015 M citrate (Na+, pH 6.8, 10 hr), and exhaustive dialysis cs. H2G. Gel Filtration of tRNA. A column of Sephadex G-100 (93 X 0.9 cm) at 22" was equilibrated and eluted with 0.15 M KCI-0.01 M MgC12-0.0005 M EDTA-0.01 M cacodylate (Na+, pH 7.0); 10-min fractions (1.8 ml) were collected and A 2 6 0 was determined directly. Radioactivity was determined by counting 1-ml aliquots. Alkaline Hydrolj.sis of tRNA. tRNA (1.0-1.5 mg/ml) in 0.3 M KOH (3-4.5 ml) was incubated at 37" for 18 hr in a sealed polyethylene tube. The digestion mixture was adjusted to pH 7 with formic acid, diluted 20-fold with H 2 0 , and applied to a Dowex AG 1-X4 (formate) column (25 X 0.9 cm). After a rinse with 20 ml of HeO, the column was eluted at 0.5 ml/min with two linear gradients of formic acid (HyOto 1.0 M formic acid, total volume 150 ml; 1.0 M formic acid to 4.0 M formic acid, total volume 400 ml) followed by 4.0 M formic acid (80-120 ml) and finally 2 M "21. Radioactivity
LOCATING
was determined by counting aliquots (250 111) of fractions. (Formic acid did not decrease the counting efficiency by more than 5 %.) Aliquots containing HCI were either taken to dryness or neutralized with N H 4 0 H before counting. The eluted mononucleotides were freed of formic acid by lyophilization, and were identified when possible by their ultraviolet spectra in acidic and basic solution. Radioactive peaks corresponding to 2 I - and 3 '-N4-mCMP were dephosphorylated by incubation with alkaline phosphatase (10 pgiml) in 0.15 M NH4HCOs at 37" for 18 hr. Salts were then removed by lyophilization. Descending Paper Chromafogvaphy. This was performed at 22" as described under Results. Major spots were located by ultraviolet quenching, and radioactive components by counting 1-cm wide strips of the chromatogram. 4-ThioU Content o f tRNA. This was determined from the difference spectrum (300-360 mp) between nuclease digests of untreated E. coli B tRNA and nuclease digests of E. coli B tRNA whose 4-thioU had been removed (tRNA,(,,-). tRNAIo,- was prepared by incubating tRNA (2.5 mg/ml) in 0.01 M NaIOl for 45 min at 40". The tRNA was freed of periodate by repeated precipitation with NaCl and ethanol, then dialyzed (4") first cs. 0.01 M acetate (Na+), pH 4.5 (500 volumes, 2 hr), then exhaustively cs. HyO, and finally lyophilized. Nuclease digests were prepared by incubating tRNA (1.5 mgiml) at 37' with both RNase A (10 pg/ml) and RNase T-1 (6 Fg/ml) in 0.01 M Tris-HC1 (pH 7.2). After 12 hr, the pH was adjusted to neutrality with several microliters of 1 M NaOH, and the incubation was continued for an additional 6 hr. Final digests of both tRNA and tRNAio,- exhibited hyperchromic changes of 32% at 260 mp relative to the Mg2+-containing starting polymers, indicating digestion to very short oligonucleotides. While the spectrum of the digested tRNA revealed the 4-thioUMP absorption band with maximum at 330 mp (A,30-digestedtRNA = 2.13 A n FMg*+ o containing undigested tRNA), this band was absent from the spectrum of the digest of tRNAlo,-. The only absorption at 330 mp observed with the latter was contributed by the tail of the (main) 260-mp absorption band (Aaao-digestedtRNAI,,,= 0.40z A260 Mg2+-containing undigested tRNArn,-). The difference between these two spectra was a spectrum characteristic of 4-thioUMP, i.e., it was symmetrical and gaussian between 300 and 360 mH, with a maximum at 332 mp equal to 1.75% of A260 of the Mg2+-containingundigested tRNA. Using the extinction coefficient €331 = 21.5 X l o 3(determined at pH 6.5 for 4-thiouridine (Kochetkov et al., 1963)) this absorption difference is equivalent to 0.45 mole of 4-thioU/mole of tRNA. Sensiticitj. of' Major Bases to Dericatization. Individual 2 '-deoxyribonucleosides (concentration indicated in Table 11) in 12 ml of the reagent solution used to derivatize tRNA
~ - T H I O U R I D Y L A T EI N
~ R N AS E Q U E N C E S
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DIOCHEMISI K Y
4
E 0.7
t
0.6-
5
0
0.3
w 5
N
-1000
MINUS IO4I
30
60
9
120
MINUTES
FIGURE 1 : Kinetics of incorporation of l4cfrom methylamine into E, coli B tRNA. Standard derivatization conditions were employed,
0 0 - - --,
